The present invention relates to the field of animal husbandry. More specifically it relates to novel methods of predicting and altering male fertility. It presents four novel seminal plasma proteins involved in the fertility of bulls. These proteins may have a variety of applications including use in fertility prediction and fertility alteration. The present invention describing the alteration of male fertility by contacting semen with seminal plasma of different characteristic may prove especially useful in man, either to improve male fertility with artificial insemination, or to decrease male fertility when used as or with contraceptive agents or devices.
Males with suboptimal fertility are a concern in humans as well as agricultural animals. Although males typically labeled as subfertile or infertile are characterized by poor sperm motility, high numbers of abnormal sperm or inadequate freezability, the subfertile male with normal semen parameters remains an enigma. For these males, the factors contributing to their subfertility are largely unknown.
Collectively, the various studies comparing the effects of seminal plasma on the fertility of caudal or ejaculated sperm are inconclusive. Mouse and rabbit sperm have been reported to become more fertile after addition of a low molecular weight factor from human seminal plasma [Gaur R. D., Talwar G. P., 1975, Further studies on the fertility promoting factor from human seminal plasma. Int. J. Fertil.; Vol. 20, pp 133-136]. Seminal plasma components, especially those from the seminal vesicles, have been found to augment fertility in the rat [Curry, P. T. and R. W. Atherton, 1990, Seminal vesicles: development, secretory products, and fertility. Arch. Androl. Vol. 25, pp 107-113]. Changes in seminal plasma composition resulting from removal of some or all of the accessory sex glands, also may be detrimental to fertility in the rat [Queen, K., C. B. Dhabuwala and C. G. Pierrepoint, 1981, The effect of the removal of the various accessory sex glands on the fertility of male rats. J. Reprod. Fert. Vol. 62, pp 423-426], hamster [Chow, P. H., S. F. Pang, K. W. Ng and T. M. Wong, 1986, Fertility, fecundity, sex ratio and the accessory sex glands in male golden hamsters. Int. J. of Androl. Vol. 9, pp 312-320]and mouse [Pang, S. F., P. H. Chow and T. M. Wong, 1979, The role of the seminal vesicle, coagulating glands and prostate glands on the fertility and fecundity of mice. J. Reprod. Fertil. Vol. 56, pp 129].
In contrast, several studies have concluded that sperm exposure to seminal plasma depresses sperm motility in the boar [Iwamoto, T., A. Tsang, M. Luterman, J. Dickson, E. deLamirande, M. Okuno, H. Mohri and C. Gagnon, 1992, Purification and characterization of a sperm motility-dynein ATPase inhibitor from boar seminal plasma. Molec. Reprod. and Devel. Vol. 31, pp 55-62], human [Iwamoto, T. and C. Gagnon, 1988, Purification and characterization of a sperm motility inhibitor in human seminal plasma. J. Androl. Vol. 9, pp 377-383], bull, ram, goat [Dott, H. M., R. A. P. Harrison and G. C. A. Foster, 1979, The maintenance of motility and the surface properties of epididymal spermatozoa from bull, rabbit and ram in homologous seminal and epididymal plasma. J. Reprod. Fert. Vol. 55, pp 113-124], stallion, and rabbit [Corteel J. M., 1980, Essets du Plasma Seminal sur la Survie et la Fertilite des Spermatozoides Conserves In Vitro. Reprod. Nutr. Develop. Vol. 20, pp 1111-1123]. Decreases in sperm fertility after exposure to seminal plasma components also have been reported for humans [Andhya et al., 1987, Purification and partial chemical characterization of a glycoprotein with antifertility activity from human seminal plasma. Biol. Reprod. Vol. 36, pp 511-521] and rabbits [Chang, M. C., 1957, A detrimental effect of seminal plasma on the fertilizing capacity of sperm. Nature. Vol. 179, pp 258-259].
Studies evaluating the seminal plasma characteristics of individuals and their relationship to fertility are limited. When compared to fertile men, infertile men have been reported to be missing seminal plasma proteins resolved by two dimensional gel electrophoresis [Sadowski, T. and B. J. Rogers, 1985, Two-dimensional electrophoetic patterns of seminal plasma proteins from fertile and infertile men. Biol. Reprod. Suppl. 1. Vol. 32, Abstr.#130]. Distribution of some heparin binding protein have been found to differ between seminal plasma of high and low fertility bulls [Kandell, R. L., M. E. Bellin, H. E. Hawkins and R. L. Ax, 1992, Bull fertility related to distributions of heparin binding proteins in sperm membranes and seminal plasma. J. Androl. Suppl. Abst. #51]. The presence, absence or the critical concentration of proteins in seminal plasma could potentially be responsible for the effects of seminal plasma on sperm fertility.
One difficulty with previous studies attempting to establish an effect of seminal plasma on animal fertility has been the lack of good fertility data on the individuals in question. In addition, relatively small effects of seminal plasma on fertility may escape detection because of assay insensitivity. The studies described in this patent application overcome these potential problems by using cull dairy bull sires with reliable fertility data, and by using a heterospermic zona-free bovine oocyte penetration assay which negates the effect of egg variation and facilitates the detection of small differences in fertility. The goal of these studies was to determine if the in vitro fertility of ejaculated sperm could be altered by exposure to seminal plasma from a bull of contrasting fertility. In addition the possibilities of predicting male fertility based on studies of seminal plasma proteins were examined and resulted in developing a method allowing for such prediction.
Dairying is the major agricultural enterprise. A major factor affecting the success of dairying has been the increased milk production per cow which has resulted from the artificial insemination (AI) of cows with semen from genetically superior sires. The average doubling of milk production per cow over the last 40 years is largely attributable to the genetic selection and breeding services provided by the AI industry.
Reproduction is a vital factor in determining the profitability of a dairy herd. Successful reproduction not only produces a calf which may be used to replace older less productive cows in the herd, but it also ensures that the cow will continue to produce milk. A cow will only begin to lactate effectively after calving.
The ideal time interval between calves for cows can vary depending on climate and other factors. If this interval is prolonged because cows fail to conceive during the estrous cycle (about 21 days for each failed conception), it costs farmers in feed, lost milk production and decreased milk yields.
A significant factor contributing to the reproductive efficiency of a dairy cow herd is the fertility of the semen used to inseminate the cow. The AI industry is able to reliably assess the fertility of the semen of individual bulls, because relatively few bulls are used to breed large numbers of cows. The fertility value involves an estimate of the percentage of cows becoming pregnant after being bred with semen from a particular bull. Sires currently in use for AI in this country range in fertility from 55 to 80%. The use of semen from bulls of lower fertility results in a longer than average calving interval for the cow herd, which is more costly to the farmer. It is estimated that using lower fertility bulls results in a minimal loss of 500 lbs of milk production per year per cow, as well as the cost of feeding for non-productive animals.
The reasons the AI industry does not eliminate the lower fertility bulls is due to economics. Currently the AI industry has no method for determining if a bull is subfertile until after the investment of $100,000 or more has been made for the purchase, raising, and maintenance of the bull for 3-4 years until his genetic worth is known. The industry is quite competitive, and semen marketing focuses on the genetic characteristics of milk production of the daughters sired by the bull. If a bull produces daughters with highly desirable genetic characteristics for producing milk, semen from that bull will be in demand. These "high demand" bulls are what those in the AI industry hope to discover, because they can make the difference between a profit or loss for the entire company.
Therefore, once a genetically superior sire has been identified it will only be eliminated from the bulls providing semen for AI if its fertility is extremely poor. Bulls that are 5-15% below average fertility or "subfertile" will still be retained if the characteristics of their daughters are in demand.
The applicants invention presents some new ways of solving the problems outlined above. It provides a novel method for predicting fertility based on the testing of the protein make-up of seminal plasma. It also provides a method for improving fertility of sperm obtained from low fertility bulls. The latter procedure involves harvesting seminal plasma from very high fertility bulls, and using it, or components of it, to treat sperm from bulls with average or below average fertility. The native seminal plasma is removed from the sperm, which is then resuspended in seminal plasma or other medium supplemented with components thereof, obtained from high fertility bulls. The four major seminal plasma proteins involved in the fertility of semen have been identified. The instant invention allows for improvement of fertility of lower fertile but high demand bulls with the seminal plasma or component through obtained from high fertility bulls, which are not necessarily high demand animals.
By the way of background, semen consists of both sperm and seminal plasma. Male fertility is influenced by inherited factors directly associated with the sperm. Reports for several species suggest that seminal plasma contains factors which also influence male fertility. These studies were generally based on comparisons of seminal plasma composition between males of differing fertility [Constentino M. J., Emilson L. B. V., Cockett A. T. K., 1984, Prostaglandins in semen and their relationship to male fertility: A study of 145 men. Fertil Steril; Vol. 41, pp 88-94, Sandowski T., Rogers B. J., 1985, Two-dimensional electrophoretic patterns of seminal plasma proteins from fertile and infertile men. Biol Reprod, Vol. 32, (suppl 1), p 102, Jeyendran R. S., Van der vern H. H., Rosecrans R., Perez-Pelaez M., Al-Hasani S., Zaneveld L. J. D., 1989, Chemical constituents of human seminal plasma: Relationship to fertility. Andrologia, Vol. 21, pp 423-428, Panidis D., Rousso D., Pappas C., Kalogeropoulos A., 1991, Seminal plasma transferfin: does it help in the diagnosis of fertility. J. Obst. Gyn., Vol. 11, pp 211-214, Autiero M., Sansone G., Abreccia P., 1991, Relative ratios of lactoferrin. albumin, and acid phosphatase seminal levels as sperm quality markers in fertile and infertile men. J. Androl., Vol. 12, pp 191-200, Kandell R. L., Bellin M. E., Hajokins H. E., Ax R. L., 1992, Bull fertility was related to distribution of heparin binding proteins in sperm membrane and seminal plasma. J. Androl., Vol. 13(suppl 1), p 30, ] or the isolation of factors from seminal plasma which facilitate or inhibit sperm capacitation, fertilization, and related events [Dukelow W. R., Cheinoff H. N., Williams W. L., 1967, Properties of decapacitation factor and presence on various species. J. Reprod. Fert., Vol. 14, pp 393-399; Hunter A. G., Nornes H. O., 1969, Characterization and isolation of a sperm coating antigen from rabbit seminal plasma with capacity to block fertilization. J. Reprod. Fertil. Vol. 20, pp 419-427, Eng L. E., Oliphant G., 1978, Rabbit sperm reversible decapacitation by membrane stabilization with a highly purified glycoprotein from seminal plasma. Biol. Reprod., Vol. 19, pp 1083-1094, Reddy J. M., Stark R. A., Zaneveld L. J. D., 1979, A high molecular weight antifertility factor from human seminal plasma. J. Reprod. Fert.; Vol. 57, pp 437-446, Gaur R. D., Talwar G. P., 1975, Further studies on the fertility promoting factor from human seminal plasma. Int. J. Fertil., Vol. 20, pp133-136, Shivaji S., Bhargava P. M., 1987, Antifertility factors of mammalian seminal fluid. BioAssays, Vol. 7 pp 13-17, Audhya T., Reddy J., Zaneveld L. J. D., 1987, Purification and partial chemical characterization of a glycoprotein with antifertility activity from human seminal plasma. Biol. Reprod. Vol. 36, pp 511-521, Miller D. J., Winer M. A., Ax R. L., 1990, Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin. Biol. Reprod., Vol. 42, pp 899-915].
Antifertility factors from seminal plasma have been described for several species and include decapacitation factors purified to various degrees [Shivaji S., Scheit K. H., Bhargava P. J., 1990, Proteins of Seminal Plasma. New York: John Wiley and Sons, Inc.; pp 332-33, ] human antifertility factor 1 (AFl) [Reddy J. M., Stark R. A., Zaneveld L. J. D., 1979, A high molecular weight antifertility factor from human seminal plasma. J. Reprod. Fert., Vol. 57, pp 437-446, Audhya T., Reddy J., Zaneveld L. J. D., 1987, Purification and partial chemical characterization of a glycoprotein with antifertility activity from human seminal plasma. Biol. Reprod.; Vol. 36, pp 511-521, Reddy J., Audhya T., Goodpasture J. C., Zaneveld L. J. D., 1982, Properties of a highly purified antifertility factor from human seminal plasma. Bio. Reprod., Vol. 27, pp 1076-1083, ], rabbit acrosome stabilizing factor (ASF) [Eng L. E., Oliphant G., 1978, Rabbit sperm reversible decapacitation by membrane stabilization with a highly purified glycoprotein from seminal plasma. Biol. Reprod., Vol. 19, pp 1083-1094, ] and bull seminal plasmin (SPLN) [Shivaji S., Bhargava P. M., 1987, Antifertility factors of mammalian seminal fluid. BioAssays, Vol. 7, pp 13-17, ]. Generally, these factors are believed to inhibit sperm capacitation, the acrosome reaction or acrosomal enzymes and are believed to ultimately interfere with fertilization.
Recent studies [Miller D. J., Winer M. A., Ax R. L., 1990, Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin. Biol. Reprod., Vol. 42, pp 899-915, Nass S. J., Miller D. J., Winer M. A., Ax R. L., 1990, Male accessory sex glands produce heparin-binding proteins which bind to cauda epididymal spermatozoa and are testosterone-dependent. Mol. Reprod. Dev., Vol. 25, pp 237-246] have suggested that heparin-binding proteins in bull seminal plasma are taken up by cauda epididymal sperm membranes. Because other studies have indicated that the ability of sperm to bind heparin and other glycosaminoglycans is correlated with semen quality and fertility [Ax R. L., Lenz R. W., 1987, Glycosaminoglycans as probes to monitor differences in fertility of bulls. J. Dairy Sci., Vol. 70, pp 1477-1486, Vasquez J. M., Winer M. A., Ax R. L., Boone W. R., 1989, Correlation of human spermatozoa heparin binding with the zona-free hamster egg in vitro penetration assay. Am. J. Obstet. Gynecol., Vol. 160, pp 20-26, Blottner S., Nehring H., Torner H., 1990, Individual differences in capacitation of bull spermatozoa by heparin in vitro. Relationship to Fertility. Theriogenology, Vol. 34, pp 619-628], heparin- binding proteins in seminal plasma may positively influence fertility.
The applicants developed an interest in this topic after surveying the fertility of reproductively "normal" dairy bulls primarily housed at artificial breeding cooperatives in Pennsylvania and New York. In vivo fertility data summarized for 131 bulls with normal semen parameters, as assessed by laboratory evaluation, indicated that the fertility of the bull population followed a distribution ranging from 8.5 below to 5.4 percentage points above an average fertility designated as zero (FIG. 1). This relatively small range of fertility differences among bulls in the normal population was distinguishable because fertility data were available for individual bulls based on more than 1,000 breedings by artificial insemination. The applicants are not aware of any other species for which such data are available on individual sires. These circumstances provide a unique animal model for the study of factors contributing to the relative fertility of the normal male.
Current genetic selection criteria of dairy as well as beef bull sires is based largely on milk and beef production of the offsprings. Genetic selection does not focus on bull fertility due to the lack of a method to predict bull fertility prior to the obtaining of the second generation data. This practice results in a wide range of fertilities of dairy and beef sires and significant losses to the farmers and breeders due to inefficient reproduction. The presented invention will significantly raise fertility of semen being sold for artificial insemination, without affecting current genetic selection practices.